The present invention relates to the field of apoptosis, as well as to the field of cancer diagnosis and treatment, and treatment and diagnosis of auto-immune diseases and other diseases by induction of apoptosis. In particular the invention provides novel molecules and means to induce apoptosis or enhance apoptosis. The novel molecules and means are part of the apoptotic pathway induced by apoptin. Apoptin is a protein originally found in chicken anemia virus (CAV; Noteborn et al., 1991) and was originally called VP3. The apoptotic activity of this protein was discovered by the group of the present inventors (Noteborn et al., 1994).
As stated above the present invention makes use of the induction of apoptosis in which Bip-like proteins are involved.
Apoptosis is an active and programmed physiological process for eliminating superfluous, altered or malignant cells (Earnshaw, 1995, Duke et al., 1996). Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus, condensation and cleavage of DNA into domain-sized fragments, in most cells followed by internucleosomal degradation. The apoptotic cells fragment into membrane-enclosed apoptotic bodies. Finally, neighbouring cells and/or macrophages will rapidly phagocytose these dying cells (Wyllie et al., 1980, White, 1996). Cells grown under tissue-culture conditions and cells from tissue material can be analysed for being apoptotic with agents staining DNA, as e.g. DAPI, which stains normal DNA strongly and regularly, whereas apoptotic DNA is stained weakly and/or irregularly (Noteborn et al., 1994, Telford et al., 1992).
The apoptotic process can be initiated by a variety of regulatory stimuli (Wyllie, 1995, White 1996, Levine, 1997). Changes in the cell survival rate play an important role in human pathogenesis, e.g. in cancer development, which is caused by enhanced proliferation but also by decreased cell death (Kerr et al., 1994, Paulovich, 1997). A variety of chemotherapeutic compounds and radiation have been demonstrated to induce apoptosis in tumor cells, in many instances via wild-type p53 protein (Thompson, 1995, Bellamy et al., 1995, Steller, 1995, McDonell et al., 1995).
Many tumors, however, acquire a mutation in p53 during their development, often correlating with poor response to cancer therapy. Transforming genes of tumorigenic DNA viruses inactivate p53 by directly binding to it (Teodoro, 1997). An example of such an agent is the large T antigen of the tumor DNA virus SV40. For several (leukemic) tumors, a high expression level of the proto-oncogene Bcl-2 or Bcr-abl is associated with a strong resistance to various apoptosis-inducing chemotherapeutic agents (Hockenberry 1994, Sachs and Lotem, 1997).
For such cancers (representing more than half of the tumors) alternative anti-tumor therapies are under development based on induction of apoptosis independent of p53 (Thompson 1995, Paulovich et al., 1997). One has to search for the factors involved in induction of apoptosis, which do not need p53 and/or can not be blocked by Bcl-2/Bcr-abl-like anti-apoptotic activities. These factors might be part of a distinct apoptosis pathway or being (far) downstream to the apoptosis inhibiting compounds.
Apoptin is a small protein derived from chicken anemia virus (CAV; Noteborn and De Boer, 1995, Noteborn et al., 1991, Noteborn et al., 1994), which can induce apoptosis in human malignant and transformed cell lines, but not in untransformed human cell lines. In vitro, apoptin fails to induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial and smooth-muscle cells. However, when normal cells are transformed they become susceptible to apoptosis by apoptin. (Danen-van Ooschot, 1997 and Noteborn, 1996). Long-term expression of apoptin in normal human fibroblasts revealed that apoptin has no toxic or transforming activity in these cells.
In normal cells, apoptin was found predominantly in the cytoplasm, whereas in transformed or malignant cells i.e. characterized by hyperplasia, metaplasia or dysplasia, it was located in the nucleus, suggesting that the localization of apoptin is related to its activity (Danen-van Oorschot et al. 1997).
Apoptin-induced apoptosis occurs in the absence of functional p53 (Zhuang et al., 1995a), and cannot be blocked by Bcl-2, Bcr-abl (Zhuang et al., 1995), the Bcl-2-associating protein BAG-1 and not by the caspase-inhibitor cowpox protein CrmA (Danen-Van Oorschot, 1997a, Noteborn, 1996).
Therefore, apoptin is a potent agent for the destruction of tumor cells, or other hyperplasia, metaplasia or dysplasia which have become resistant to (chemo)therapeutic induction of apoptosis, due to the lack of functional p53 and (over)xe2x80x94expression of Bcl-2 and other apoptosis-inhibiting agents. (Noteborn et al., 1997).
The fact that apoptin does not induce apoptosis in normal human cells, at least not in vitro, suggests that a toxic effect of apoptin treatment in vivo will be very low. Noteborn et al. (1997) have provided evidence that adenovirus expressed apoptin does not have an acute toxic effect in vivo. In addition, in nude mice it was shown that apoptin has a strong anti-tumor activity.
It appears, that even pre-malignant, minimally transformed cells, may be sensitive to the death-inducing effect of apoptin. In addition, Noteborn and Zhang (1997) have shown that apoptin-induced apoptosis can be used as diagnosis of cancer-prone cells and treatment of cancer-prone cells.
Knowing that apoptin is quite safe in normal cells, but that, as soon as, a cell becomes transformed and/or immortalized (the terms may be used interchangeable herein) the present inventors designed novel means and methods for the induction of apoptosis based on the identification of compounds involved in the apoptin-induced apoptotic cascade. These compounds are factors of an apoptosis pathway, which is specific for transformed cells. Therefore, these proteins are very important compounds in new treatments and diagnosis for diseases related with aberrancies in the apoptotic process, such as cancer, and auto-immune diseases.
A group of proteins found to be associated with the apoptotic pathway is the family of Bip-like proteins.
Thus the invention provides a recombinant and/or isolated nucleic acid molecule encoding at least a functional part of a member of the family of Bip/GRP78-like proteins comprising at least a functional and/or specific part of the sequence given in FIGS. 1, 2, 3, 4 or 5 or a sequence at least 70%, preferably 80, most preferably 90% homologous therewith. In one possible mechanism of action Bip-like proteins which are chaperones bind to apoptin or apoptin-like proteins resulting in a conformational change in the apoptin-like proteins resulting in enhanced apoptotic activity. Protein-like activity herein is defined as any molecule indirectly or directly providing similar activity as the original protein (in kind not necessarily in amount). It is preferred to bring the Bip-like activity into a cell, which can be done suitably using an expression vector. It is of course preferred if not required that such a cell is also provided with apoptotic, preferably apoptin-like activity. A very suitable manner is to provide apoptin-like activity on another or the same vector.
The invention also provides a recombinant and/or isolated proteinaceous substance having Bip/GRP78-like activity and comprising at least a functional part of the sequence of FIG. 6 or FIG. 7 or a functional equivalent thereof or being encoded by a nucleic acid molecule according to claim 1. Except as being used for enhancing apoptosis this proteinaceous substance can also be used to identify further apoptotic agents. Such agents are therefor also part of the present invention.
The invention further provides a method for inducing apoptosis in a cell comprising providing said cell with Bip/GRP78 inhibiting activity, preferably together with apoptin-like activity.
As stated the invention provides a method for inducing apoptosis through interference with the function of Bip-like proteins (interchangeably referred to as Bip or Bip-like proteins).
The invention provides an anti-tumor therapy based on the interference with the function of Bip-like proteins. The fact that Bip-like proteins are abundantly present in tumor cells in combination with highly-expressed oncogenes and that Bip associates with apoptin, makes Bip-like proteins very important targets of an anti-tumor agent.
The invention provides Bip as the mediator of apoptin-induced apoptosis, which is tumor-specific.
The invention will be explained in more detail in the following experimental part. This only serves for the purpose of illustration and should not be interpreted as a limitation of the scope of the invention.